Systems-level analysis provides insights on methanol-based production of L-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus
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Background
Bacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain B. methanolicus MGA3 naturally overproduces l -glutamate in methanol-based fed-batch fermentations.
Results
Here we generated, by induced mutagenesis, an evolved B. methanolicus strain exhibiting enhanced l -glutamate production capability (>150%). To showcase the potential of this evolved strain, further metabolic engineering enabled the production of γ-aminobutyric acid (GABA) directly from l -glutamate, with a yield of 13.2 g/L from methanol during fed-batch fermentations. By using a systems-level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and genome-scale metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards α-ketoglutarate and its derivative, l -glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on l -glutamate production, likely due to an upregulation of various transaminases involved in the l -glutamate biosynthesis from α-ketoglutarate.
Conclusions
These results and insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of l -glutamate and its derivatives.
